EMBO/soda-nel huggingface dataset:
The Mutational Landscape of the SCAN-B Real-World Primary Breast Cancer Transcriptome (2020)
Figure 7

Fig 7: Impact of tumor mutational burden on overall survival across treatment groups

Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.
Frequency modulation of ERK activation dynamics rewires cell fate. (2015)
Figure 1

Sustained <styled-content style="fixed-case">GF</styled-content> stimulation induces heterogeneous <styled-content style="fixed-case">ERK</styled-content> activity dynamics

EKAR2G biosensor. Upon phosphorylation by ERK, binding of the WW phospho‐recognition domain to the phosphorylated Cdc25 substrate sequence leads to spatial re‐orientation of donor (mTFP1) and acceptor (Venus) fluorophores leading to a FRET change that can be ratiometrically measured.

Ratiometric and mTFP1 donor images of EKAR2G in an EGF‐stimulated PC‐12 cell at the indicated time points, with t = 0′ corresponding to EGF application. Upper panel: FRET ratio image is color‐coded for ERK activity. Lower panel: raw mTFP1 donor image in black/white contrast. Scale bar = 20 μm.

Flow‐based, microfluidic device for temporal GF delivery. Computer‐controlled, pressure pump enables mixing of medium and GFs in the control part (left), and temporally defined GF delivery in the cell culture chamber (right and magnified inset).

Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody.

Population average of ERK activation dynamics cell‐averaged EKAR2G emission ratios (ERs) from n= at least 111 cells. StDev are shown.

Selected EKAR2G ratio time series illustrating, from top to bottom, sustained, oscillatory, or transient ERK activity dynamics. ER is color‐coded as in bar. Scale bar = 20 μm.

Single‐cell ERK activity trajectories. Cell‐averaged ERs for n = 10 cells, standard deviation range (StDev), population average for the indicated GFs and dosages. Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application.

Waterfall plots of single‐cell ERK activity trajectories. Cell‐averaged ER trajectories are color‐coded (n = 78 cells), population average (bottom). Vertical dotted line indicates GF application.

Source data are available online for this figure.
Frequency modulation of ERK activation dynamics rewires cell fate. (2015)
Figure 2

Quantification of <styled-content style="fixed-case">ERK</styled-content> activity trajectory heterogeneity

Trajectories from all sustained GF stimulation experiments (n = 307 cells, same ERK activation trajectories as in Fig 1) were pooled and five representative trajectories were identified using k‐means clustering with squared Euclidean distance. Raw (color‐coded by cluster) and cluster representative trajectories (black).

Overlaid cluster representative trajectories.

Population distribution of representative ERK activity trajectories in response to different GF dosages. Data representative of n = 3 experiments.

Peak emission ratio intensity for each cluster. Boxplots with median, interquartile (box) and 1.5 IQR (whiskers) range, and raw datasets are shown. Boxplot notches extend 1.58 IQR / √Nobs, which gives approximately 95% confidence interval for comparing medians.
Frequency modulation of ERK activation dynamics rewires cell fate. (2015)
Figure 3

<styled-content style="fixed-case">ERK</styled-content> activity responses to single <styled-content style="fixed-case">GF</styled-content> pulses

A–D

Single GF pulse experiments. Cell‐averaged ERs, population average, and StDev range for n = 10 cells. Pulse application indicated by black bars. 3′ (A, C) and 10′ (B, D) pulse. High (A, B) and low (C, D) GF concentrations.

E

Rationale for measuring amplitude and duration of 1st
ERK activity peak in response to GF stimulation. Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs. Peak duration was estimated as the time between the first point before reaching the half‐maximum of the peak in the ascending phase, and the first point after the half‐maximum in the descending phase.

F

Amplitude of 1st
ERK activity peak in response to different sustained or pulsed GF stimulation experiments. Notched boxplots of 1st
ERK activity peak amplitude with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).

G

Duration of 1st
ERK activity peak in response to sustained and pulsed GF stimulation. Boxplots of 1st
ERK activity peak duration with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).

Source data are available online for this figure.
Frequency modulation of ERK activation dynamics rewires cell fate. (2015)
Figure 4

<styled-content style="fixed-case">ERK</styled-content> activity responses to multiple <styled-content style="fixed-case">GF</styled-content> pulses

A–C

Double‐pulse GF experiments. (A) 3′ GF/3′ pause/3′ GF. (B) 3′ GF/10′ pause/3′ GF. (C) 3′ GF/20′ pause/3′ GF. Cell‐averaged ERs, population average, and StDev range for n = 10 cells.

D–G

Multi‐pulse GF experiments. Population average ERs (n= at least 30 cells) for different EGF/NGF dosages. (D) 3′ GF/3′pause. (E) 3′ GF/10′ pause. (F) 3′ GF/20′ pause. (G) 3′ GF/60′ pause.

H

Decay kinetics of ERK activity maxima from (D–G). Boxplots with median, interquartile (box), and 1.5 IQR (whiskers) range are shown for at least n = 30 cells.

Source data are available online for this figure.
Constricted migration is associated with stable 3D genome structure differences in cancer cells (2022)
Figure 1

Figure 1. A375 cells exhibit morphological differences and increased migration efficiency after sequential rounds of constricted migration.

(A) Layout of sequential Transwell migration experiment with A375 cells.

(B) Migration efficiency (% of cells that migrated through filter pores in each round) of A375 cells during 5 μm constricted migration and 12 μm migration (left panels). Biological replicates shown. Phase contrast imaging of the final cell populations after the 10 rounds of migration, cells cultured on 2D plastic (right panels, Scale bar = 50 µm).

(C) A375 nuclei stained with DAPI (blue), Lamin A/C (green) and H3K9me3 (red) in the indicated migration conditions. Images shown represent a maximum projection of z-stacks (Scale bar= 5 µm).

(D) Cumulative distribution plot of Lamin A/C coefficient of variation in all indicated A375 conditions (Control (n = 59), Top-5 (n = 64), Bottom-5 (n = 62) nuclei). Kolmogorov-Smirnov test p-values indicated in plot.

(E) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control (n = 59), Top-5 (n = 63), Bottom-5 (n = 64) and Bottom-12 (n = 80) nuclei (** p = 0.0069, two-tailed t-test). Error bars = mean ± SD.

(F) Maximum projection confocal images of Top-5 and Bottom-5 cells embedded in collagen and stained with DAPI (blue) and Phalloidin (red). (Inset) Zoom in of nucleus of Bottom-5 cell. Top-5 and Bottom-5 zoomed inset scale = 5 μm; Bottom-5 zoomed out image scale = 20 μm.

(G) Fraction of Control, Top-5 and Bottom-5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices. Each biological replicate is connected by a line.

(H) Nuclei of Top-5 (left) and Bottom-5 cells (right) embedded in 3D collagen matrix and stained with DAPI (blue) and Lamin A/C (green). Scale = 5μm. For 3D reconstruction visualization, see Movies EV5 and EV6.

(I) Percent of cells embedded in collagen that had elongated nuclei (Aspect Ratio > 2.5) for A375 Control (n = 54), Top-5 (n = 52) and Bottom-5 (n = 53).

(J) Cumulative distribution plot of Lamin A/C coefficient of variation for Control (n = 70), Top-5 (n = 78) and Bottom-5 (n = 109) cells embedded in 3D collagen matrices. Kolmogorov-Smirnov test p-values indicated in plot.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 1

Figure 1. 2P-FLIM reveals nutrient control of neuronal mitochondrial activity.

A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos).

B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%).

C, D a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C) and human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 2

Figure 2. Nutrient-mediated regulation of mitochondrial activity in cultured neurons depends on mTORC1.

A, B Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A) and human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test.

C Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 3

Figure 3. Lysosomal mTORC1 regulates mitochondrial activity in cultured neurons and in brain.

A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test.

B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m.

C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 4

Figure 4. Lysosomal, but not mitochondrial mTORC1 controls NiMA.

A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 hours, and then imaged by 2P-FLIM before and 30 minutes after addition of insulin. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of 3 independent assays.

B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays.

C Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 5

Figure 5. NiMA control of mitochondrial DNA replication in neurons is disrupted by AβOs.

A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2.

B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit ­mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test and are representative of 3 independent assays.

C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ± s.e.m.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 6

Figure 6. AβOs block NiMA by disrupting mTORC1 activation al lysosomes.

A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrigel (Choi et al, 2014). Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test.

B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor-Rheb15 expression was confirmed by western blot­ting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 7

Figure 7. AβO-mediated inhibition of NiMA requires tau.

A Cortical mouse neurons from tau knockout (KO) mice were serum-starved in Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin or amino acids were immediately added, and the cells were then imaged again 30 minutes later.

B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neurons (see Figure 6B).

Data information: Each colored line in (A) and (B) refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test.
A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-β Oligomers (2018)
Figure 8

Figure 8. NiMA is dysregulated in tuberous sclerosis fibroblasts.

A-C Wild type mouse cortical neurons depleted of Nprl3 or Tsc2 with shRNA had mitochondria that were insensitive to amino acids (R+L) or insulin, respectively. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test.

D, E (D) Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin. (E)

Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immun­o­fluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test.
Miro1 dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity (2016)
Figure 3

Figure 3. Vesicular release is reduced in the presence of mitochondria.

A, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.
Miro1 dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity (2016)
Figure 4

Figure 4. Neuronal activity influences mitochondrial occupancy of presynaptic terminals in a Miro1-dependent manner.

A, C & E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D & F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p<0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p<0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p>0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.