EMBO/soda-nel huggingface dataset:
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 6

Figure 6. Increased expression of pgam5 by rIFN-β treatment is beneficial in 6OH-DA- and hSCNA-induced models of neurodegeneration.

A-B. Immunohistochemistry of TH in coronal sections of the striatal region in mice treated with 6OH-DA and rIFN-β and quantification.

C-D. Immunohistochemistry of TH in coronal sections of the substantia nigra in mice treated with 6OH-DA, with or without rIFN-β and quantification.

E-K. Immunofluorescence in the striatal region in mice treated with 6OH-DA and rIFN-β for optineurin (E, quantified in F) and Tom20 (E), with quantification of individual mitochondrial size by mitochondrial section area (G). 8OHdG- (H-I), OxDJ1- (J-K), and DAPI-labeled nuclei. Scale bars equal 10 microns.

L-M. PGAM5 and TH immunostaining (L) and quantification (M) in striatum from mice treated with 6OH-DA and rIFN-β.

N. Percentage of right versus left forepaw use before and 21 days after injection with hSNCA (left hemisphere) and control lentivirus (CTL) or hSNCA and lenti-Ifnb (right hemisphere). Bars are mean + SEM. YYY p<0.001 by ANOVA and **p<0.01 by post hoc Tukey's multiple comparison correction. N=6/hSCNA-CTL and 8/hSCNA-Ifnb.

O. Immunostaining of TH and human α-synuclein in SN. Scale bars equal 20 microns

P. TH immunostaining in brains of rats injected with hSNCA (left hemisphere) and control lentivirus (CTL) or hSNCA and lenti-Ifnb (right hemisphere). Scale bar equals 1 mm.

Q. Immunostaining of PGAM5 in brains of rats injected with hSCNA and control or hSCNA and Ifnb. Scale bars equal 100 microns.

R. Quantification of P.

S. Quantification of Q.

T. Correlation of levels of PGAM5 and TH in rat brains injected with hSNCA (left hemisphere) and control lentivirus (CTL) or hSNCA and lenti-Ifnb (right hemisphere) by Pearson correlation.

Data information: For all graphs, error bars mean SEM; N=4, unless otherwise stated. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.001.
Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins (2022)
Figure 1

Fig. 1: DENV NS1 binds to human high-density and low-density lipoproteins

(A) Size exclusion chromatography profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS-PAGE and N-terminal sequencing as the Apolipoprotein B scaffold of the low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.

(B) Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.

(C) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre-incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation velocity analysis.
Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins (2022)
Figure 2

Fig. 2: Analysis of NS1-HDL complexes by electron microscopy reveals the presence of NS1 dimers on the surface of HDL particles

(A, B) Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL complexes. White bar: 50 nm, Black bar: 20 nm.

(C) Fitting of the NS1 3D structure of the dimeric form into the most abundant class of NS1-HDL complexes, suggesting a collapse of the NS1 hexamer into hydrophobic dimeric blocks that float and anchor into the HDL lipid phase.

(D) Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1-HDL mixture at a 2:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.

(E) Binding inhibition of hexameric NS1 to HDL with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.
Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins (2022)
Figure 3

Fig. 3: The NS1-HDL lipoprotein complex triggers pro-inflammatory signals in human primary macrophages

(A-D) Human primary macrophages were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS buffer, LPS, mix LPS-HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D) Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.
Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins (2022)
Figure 4

Fig. 4. Different biological and virological parameters measured in human plasma.

(A-H) DENV-infected hospitalized patients from the Kampong Cham Referral Hospital, Cambodia, presented either dengue with warning signs or severe dengue. Two blood samples were recovered for each patient on the day of hospital admission and during a follow-up visit that occurred before discharge from the hospital (on average 4 days apart). (A) Number of patient samples tested over the hospitalization period for their levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB complexes in addition to (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. NS1-ApoA-I and NS1-ApoB complexes are representative of NS1-HDL and NS1-LDL complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.

(I) Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE; The Native Antigen Company) were spiked in normal human plasma and NS1-ApoA-I complexes further detected by ELISA. Data represent the mean values of two technical replicates.

EXPANDED VIEW FIGURE LEGENDS
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal (2021)
Figure 1

Figure 1: A prolonged G1 arrest following CDK4/6 inhibition in RPE1 cells causes defects in the next cell cycle.

(A) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.

(B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment.

(C) Cell cycle profile of individual RPE1-FUCCI cells (each bar represents one cell) after washout from 1 (top panel), 4 (middle panel) or 7 (bottom panel) days of treatment with CDK4/6 inhibitor, at the indicated doses (same concentration used in panel B). STLC (10 μM) was added to prevent progression past the first mitosis. 50 cells were analysed at random for each repeat and 3 experimental repeats are displ­­ayed (150 cells total). Underneath the single cell profiles are quantifications of the observed cell cycle defects and G1 durations. Note, G1 length is estimated by mKO-Cdt1 expression, and G1 lengths of less than 6h could not be quantified since movies were only stated after the 6h drug washout period. These are indicated on the graphs as ≤6h. Bar graphs show mean + SD. In the violin plots, horizontal lines display the median, and error bars show 95% confidence intervals.

(D) Representative images and quantification of colony forming assays of RPE1 cells treated with CDK4/6 inhibitor for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Bar displays mean data + SEM from 3 experiments.
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal (2021)
Figure 2

Figure 2. p53 loss restores cell cycle progression and enhances long-term growth following prolonged CDK4/6 inhibition in RPE1 cells.

(A) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.

(B) Cell cycle profile of individual p53-WT or KO RPE1-FUCCI cells (each bar represents one cell) after washout from 1, 4 or 7 days of treatment with palbociclib (1.25μM). STLC (10 μM) was added to prevent progression past the first mitosis. 50 cells were analysed at random for each repeat and 3 experimental repeats are displ­­ayed (150 cells total). The right side panels show quantifications of cell cycle defects and G1 lengths from the displayed single cell profile plots. Note, G1 length is estimated by mKO-Cdt1 expression, and G1 lengths of less than 6h could not be quantified since movies were only stated after the 6h drug washout period. These are indicated on the graphs as <6h. Bar graphs show mean + SD. In the violin plots, horizontal lines display the median, and error bars show 95% confidence intervals.

(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.

(D) Cell cycle profile of individual p53-WT or KO RPE1-FUCCI cells to analyse multiple rounds of division following washout from 1, 4 or 7 days of treatment with palbociclib (1.25μM). FUCCI profiles show 50 cells analysed at random from one experiment, which is representative of 3 experimental repeats. The right side panels show quantifications of cell cycle defects and G1 lengths from the 3 experimental repeats. Note, G1 length is estimated by mKO-Cdt1 expression, and G1 lengths of less than 6h could not be quantified since movies were only stated after the 6h drug washout period. These are indicated on the graphs as <6h. Bar graphs show mean + SD. In the violin plots, horizontal lines display the median, and error bars show 95% confidence intervals.

(E) Quantification of cell cycle profiles from cells treated as in D. Graph shows the mean percentage of cells + SD that divide at each round of division, with 150 cells analysed in total from 3 experimental repeats.
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal (2021)
Figure 3

Figure 3. Prolonged CDK4/6 inhibition in RPE1 cells induces replication stress and p53-dependent cell cycle withdrawal.

(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM.

(B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals.

(C) Immunofluorescence images of DAPI and γH2AX staining in p53-WT or KO RPE1 cells either before or 48 hours after release from a 7-day treatment with palbociclib (1.25μM). Scale bar = 250 μM, zoom inserts = 3x magnification of highlighted regions.

(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.

(F) Immunofluorescence images showing symmetrical 53BP1 staining following mitotic exit in p53-KO cells after release from 7 days of palbociclib arrest. 3 separate examples are displayed. Scale bar = 25 μM

(G-I) Quantification of the first mitosis in GFP-53BP1/H2B-RFP RPE1 cells after release of from a 1-day or 7-day palbociclib (1.25μM) arrest. Quantified from the same movies are nuclear morphology after mitosis (G), chromosome segregation defects during mitosis (H) and appearance of 53BP1 foci after mitosis (I). 54 cells (1-day) or 80 cells (7-day) were analysed in total from 2 experiments. Errors bars display SD.

(J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal (2021)
Figure 6

Figure 6. Prolonged G1 arrest following palbociclib treatment in breast cancer cells downregulates replisome components, causes DNA damage, and reduces long-term proliferation.

(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C).

(B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).

(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.

(E) Cumulative mitotic entry of cells following washout from 0, 1, 4 or 7 days treated of palbociclib (1μM). 50 cells were quantified per experiments and graphs display mean -/+ SEM from 3 experiments.

(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal (2021)
Figure 7

Figure 7. Constitutive palbociclib treatment in various tumour lines causes cell cycle delays and DNA damage.

(A) Quantification of cumulative mitotic entry over time immediately following (1μM) palbociclib treatment in a range of tumour cell lines, as indicated. Mitotic entry of 50 cells per condition were analysed immediately following drug addition. Graph shows cumulative mean -/+ SD from three experiments.

(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.

(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.

Expanded view figure legends
Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress (2022)
Figure 1

Figure 1: Similar p53 dynamics can drive distinct cell fates

A) p53 expression dynamics were measured in single cells using a fluorescently labeled p53-mCherry construct in conjunction with a H2B-mCerulean nuclear marker to define the nucleus of individual cells. Images show representative single cells over time. Arrowhead highlights a single cell over multiple time points. Scale bar=20µm.

B) Single cell traces for representative cells exposed to increasing doses of H2O2 (red) or NCS (blue). Dynamics switch from pulses at low doses of damage to rising dynamics at higher doses.

C) Comparison of population averages of single cell p53 expression in response to H2O2 (red) or NCS (blue) across doses. Thick line represents mean p53 fluorescence intensity with shaded areas representing ±SD. n, represents number of individual cells analyzed per condition.

D) Bar graphs comparing integrated p53 expression over the 24-hour range for both NCS and H2O2 treatment. P-values calculated by one-way ANOVA. Data represents mean±SEM based on the number of cells analyzed for each condition (n). n for each condition is shown in Figure 1C.

E) Percentage of cell death was calculated by flow cytometry staining for Annexin V and propidium iodide for indicated doses of NCS and H2O2 24-hours post-treatment. P-values were calculated using one-way ANOVA for multiple comparisons. Data represents the mean±SEM for three biological replicates (n=3).

F) Percentage of cell death in the presence or absence of p53 knockdown after 24-hours with 300 µM H2O2 treatment. Data represents the mean±SEM for three biological replicates (n=3). Statistics performed using one-way ANOVA.

G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.

Data information: ** p<0.01, ns=not significant.
Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress (2022)
Figure 2

Figure 2. MAPKs exhibit distinct activation profiles between double strand breaks and oxidative stress

A) Schematic of hTERT-HPNE cell lines used to assess p53 and MAPK signaling. Each cell line contained an H2B-mCerulean nuclear marker, p53-mCherry, and kinase translocation reporter (KTR) for either ERK, JNK, or p38 labeled with mClover. KTR provide readout of kinase activity based on subcellular localization.

B) Images represent localization of KTR for ERK, JNK, or p38 under basal growth conditions. Scales bars=20µm.

C-E) Kinase activity for ERK (C), JNK (D), or p38 (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.

F-H) Comparison of kinase activity measured by C/N ratio for ERK (F), JNK (G), or p38 (H) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. Box plots compare C/N ratio at the observed H2O2 induced peak (dotted line). Central band represents median value, with the box bounding the 25th-75th percentiles. Whiskers extend to extreme values not considered an outlier (approximately ±2.7σ). n, for each condition and kinase is shown in the above plots and represents number of individual cells analyzed per condition. Statistics performed using one-way ANOVA. (*** p<0.001).

Data information: *** p<0.001.
Coordination of MAPK and p53 dynamics in the cellular responses to DNA damage and oxidative stress (2022)
Figure 3

Figure 3. JNK dynamics regulate induction of cell death

A) Upper panel shows population average traces of ERK, JNK, or p38 in response to 300 µM H2O2 in the presence or absence of specific inhibitors. Lower panels show boxplots with max C/N ratio for each condition. Central band represents median value, with the box bounding the 25th-75th percentiles. Whiskers extend to extreme values not considered an outlier (approximately ±2.7σ). Statistics performed using one-way ANOVA ( *p<0.05, **p<0.01, *** p<0.001, ns=not significant) n, represents number of individual cells analyzed.

B) Percentage of cell death 24-hours post-treatment with 300 µM H2O2 in the presence or absence of ERK, JNK, or p38 inhibition. Statistics performed using one-way ANOVA (**p<0.01, *** p<0.001). Data represents mean±SEM. Results based on three biological replicates (n=3)

C-E) Mean kinase activation for ERK €, JNK (D), or p38 (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.

F) Left panel has boxplot showing distribution of timing on second JNK pulse between surviving and dying cells. Central band represents median value, with the box bounding the 25th-75th percentiles. Whiskers extend to extreme values not considered an outlier (approximately ±2.7σ). Statistics performed using 2-tailed t-test (** p<0.01). Right plot shows representative single cell traces of surviving (grey) cells and dying (red) cells to highlight early JNK re-activation. Number of analyzed cells for each condition (n) is shown in panel D.

G) Percentage of cell death 24-hours post- H2O2 treatment in response to different JNK inhibition timings. Upper schematic illustrates timings of different JNK inhibition regimens. Data represents mean±SEM based on a minimum of three independent biological replicates (n=3). Statistics performed by one-way ANOVA (* p<0.05, ** p<0.01 ns=not significant).

Data information: *p<0.05, **p<0.01, *** p<0.001, ns=not significant.