EMBO/soda-nel huggingface dataset:
Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. (2015)
Figure 3

LOX inhibition, in combination with gemcitabine, extends pancreatic cancer-free survival in KPC mice

A Representative fluorescence images showing the SHG signal from cross-linked collagen in untreated, LOX antibody-treated or isotype control-treated organotypic cultures of primary fibroblasts and rat tail fibrillar collagen I.

B Chart showing the percentage of cross-linked collagen by depth, based on SHG signal, in organotypic matrices treated as indicated. Error bars indicate SEM.

C Barchart showing total cross-linked collagen in organotypic matrices treated as indicated. The experiment was performed in triplicate with three different zones imaged for 80 μm, at 2.5-μm sections each. Data are shown as total pixels ± SEM.

D Kaplan–Meier (non-parametric) survival curve showing significantly extended tumor-free survival of KPC mice treated with LOX antibody (1 mg/kg i.p., twice weekly) + gemcitabine (100 mg/kg i.p., twice weekly) (green line, n = 17), compared with gemcitabine ± isotype control antibody-treated mice (blue line, n = 22, P = 0.014) or LOX antibody alone (red line, n = 9). Treatment was initiated when mice were 70 days old (randomization was not used when recruiting the mice) and the mice were treated twice weekly. Censored mice did not develop PDAC.

E H&E-stained section of PDAC harvested from LOX + gemcitabine-treated KPC mouse. Note necrosis.

F Boxplot showing quantification of necrosis in H&E sections of PDACs harvested from these mice, as indicated. At least 30 fields of view from at least five mice per cohort were scored.

G–H H&E-stained sections of PDAC harvested from LOX + gemcitabine-treated KPC mice. Note stromal collapse (G) and marked dilation of blood vessels (H).

I Representative H&E image showing a section of PDAC harvested from a control-treated KPC mouse.
Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. (2015)
Figure 4

Enhanced leukocyte infiltration and stromal markers in LOX antibody-treated mice

A Outer left panels: Immunohistochemical analysis of macrophage infiltration (by F4/80 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner left panels: Immunohistochemical analysis of neutrophil infiltration (by MPO staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner right panels: Immunohistochemical analysis of tumor vasculature (by CD31 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Outer right panels: Immunohistochemical analysis of tenascin C expression in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice.

B–D Plots showing quantification of macrophage infiltration (B), neutrophil infiltration (C) and tumor vasculature (D) in tumors from KPC mice treated as indicated. At least 30 fields of view from at least four mice per cohort were scored, and scoring was conducted blind. P-values were calculated using Mann–Whitney U-test and median is indicated by horizontal lines.

E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC (Spearman’s rho correlation coefficient = 0.61; P < 0.0001).
A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes (2019)
Figure 1

Figure 1. Generation of slow RNAPII knock-in mutant mouse ESCs.

A Cartoon depicting the mutagenesis strategy, including the genomic target locus, as well as the structure of targeting vector. Arrows indicate location of primers used for genotyping.

B Restriction enzyme diagnostic test for the presence of the R749H mutation.

C Sequence trace of cDNA showing the presence of the heterozygous and homozygous R749H mutation.

D qRT-PCR with primers specific to both wild-type and mutant RNAPII (left panel) or to the mutant form of RNAPII (right panel), confirming that only the slow version of RNAPII is expressed in homozygous slow/slow ESCs. The sequences of the respective forward primers are shown. The "WT/slow allele" primer is complementary to the sequence in exon 14 upstream of the mutation. The "slow allele only" primer has its 3' end matching the mutated codon 749 and does not anneal to the WT DNA sequence. The mean ± SEM is plotted, n=2.
A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes (2019)
Figure 4

Figure 4. Differentiation of WT and slow ESCs along the neural lineage.

A Schematic of the neural differentiation system used in this study, indicating the relevant markers that define different stages of differentiation.

B Brightfield images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT-qPCR (Nestin and Pax6) (n=3, mean ±SEM).

C Brightfield images of aggregates.

D Brightfield images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF/FGF proliferating conditions. White arrows indicate NSCs and green arrows differentiated cells. Two small panels on the right are examples of Tuj1+ neuronal cells in slow/slow NSCs cultures.

E Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.
Human Shelterin Protein POT1 Prevents Severe Telomere Instability Induced by Homology-Directed DNA Repair (2020)
Figure 1

Figure 1. POT1-loss triggers rapid telomere elongation and a DNA damage response throughout the cell cycle

A. Schematic drawing of the POT1 gene structure and two derived polypeptides. Exon 6 that has been chosen for gene editing is encircled.

B. Schematic drawing of the repair template used for CRISPR/Cas9 mediated gene editing in HEK293E cells. Positions of primers for genotyping PCR are marked with arrows.

C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.

D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.

E. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).

F. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.

Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.
Human Shelterin Protein POT1 Prevents Severe Telomere Instability Induced by Homology-Directed DNA Repair (2020)
Figure 2

Figure 2. POT1-loss leads to accumulation of single-stranded G-rich DNA, branched DNA structures and telomere fragility

A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.

B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).

C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm.

D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph.

E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.

Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.
Human Shelterin Protein POT1 Prevents Severe Telomere Instability Induced by Homology-Directed DNA Repair (2020)
Figure 3

Figure 3. The POT1-loss phenotype is caused by deprotection of the G-rich DNA overhang

A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1.

B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis).

C. Schematic of disease-associated mutations in POT1. CTCL - cutaneous T cell lymphoma, CMM - cutaneous malignant melanoma, CLL - chronic lymphatic leukemia, CP - Coats plus syndrome.

D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).

Data information: In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT for 7 days. Expression of ectopic POT1 was induced with 1 μg/ml doxycycline (Dox) for 4 days. 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.
Human Shelterin Protein POT1 Prevents Severe Telomere Instability Induced by Homology-Directed DNA Repair (2020)
Figure 4

Figure 4. Development of 2-step QTIP uncovers substantial remodeling of the telomeric proteome upon POT1-loss

A. Schematic drawing of 2-step QTIP (improved version of the quantitative telomeric chromatin isolation protocol (QTIP)).

B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.

C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.

D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.
Human Shelterin Protein POT1 Prevents Severe Telomere Instability Induced by Homology-Directed DNA Repair (2020)
Figure 6

Figure 6. Telomere elongation and branching under POT1-loss is mediated by the HDR machinery

A. Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA (pulsed-field gel electrophoresis).

B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis).

C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph.

D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days.

E. C-overhang length analysis for clone 35 upon POT1 removal (pulsed-field gel electrophoresis, hybridization with Telo-G probe).

F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 1

Figure 1. Mitochondria are enlarged and multibranched in Ifnb-/- CNs and thalamus tissue.

A, B, and C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 24 h.

A. Projection of 3D image of Tom20. Scale bars equal 20 microns.

B. Quantification of total mitochondrial mass per neuron.

C. Mean individual mitochondria volume in Ifnb+/+ and Ifnb-/- CNs.

D, E, F, and G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb-/- mice.

D. Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 microns. Voxel size equals 0.00878908x0.00878908x0.03 microns for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 microns for Ifnb-/-. Nuclei are in grey and marked with 'N.'

E. Reconstituted axonal mitochondria. Images extracted from Movies EV1 and 2.

F. Size of individual mitochondria in cell bodies and non-myelinated and myelinated axons. N>30; error bars are SD.

G. Number of branches per mitochondrion. N>30; error bars are SD.

Data information: For all graphs, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 2

Figure 2. Deficient mitochondrial metabolism and function in Ifnb-/- CNs.

A. ATP quantification on aging from 3 days to 1 year in Ifnb+/+ and Ifnb-/- midbrains. Error bars are mean + SEM from 6 mice.

B. ATP amounts in DIV18 CNs, untreated or complemented with 30 U/mL rIFN-β. Error bars equal SEM from 5 (untreated cells) or 3 (treated cells) independent experiments.

C. ATP quantification on total mitochondrial mass in DIV6 CNs. Error bars show mean + SEM from 6 independent experiments.

D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.

F. Quantification of the respiratory control ratio (RCR), calculated as the ratio between S3 and S4o.

G. ROS levels, based on mean fluorescence intensity of DCFDA in DIV18 Ifnb+/+ and Ifnb-/- CNs, cultured with or without 30 U/mL rIFN-β. Error bars equal SEM from 3 independent experiments.

H. DCFDA quantifications in δIfnb N2A cells on treatment with the mitochondria-specific antioxidant S3qet and Mitotempo.

I. MitoSox quantification in DIV6 Ifnb+/+ and Ifnb-/- CNs. Rotenone was used as a positive control for the assay.

J. Expression of oxidative stress response genes by qPCR in DIV6 Ifnb+/+ and Ifnb-/- CNs.

K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns.

L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice.

M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb-/- mice.

N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.

Data information: For all graphs, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 3

Figure 3. IFN-β promotes the mitochondrial localization and S622 phosphorylation of Drp1

A. Immunostaining of Tom20 and Drp1 in N2As depleted of Ifnb (δIfnb) by CRISPR/Cas9 and matching nontargeting control (NTC).

B. Immunoblot of Drp1 from cell fractions of wild-type (WT), δIfnb, and NTC N2As. Purity of cytoplasmic (C) and mitochondrial fractions (M) were evaluated by immunoblotting of tubulin and Tom20, respectively.

C. Quantification of B.

D. Quantification of anti-pDrp1 immunohistostains in different regions of the brain. Images shown in Fig EV3.

E. Immunoblot of phospho-S622 Drp1 in cortices and brainstem of 3-month-old Ifnb+/+ and Ifnb-/- mice and quantification.

F. Phospho-S622-Drp1 immunoblot and quantification in Ifnb+/+ and Ifnb-/- CN cultures. Vinculin was used as a loading control. Error bars are SEM of 3 independent experiments.

G. Kinetics of Drp1 phosphorylation on S622 on rIFN-β treatment in wild-type CN cultures. Error bars are SEM from 3 independent experiments.

H. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity.

I. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb-/- CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments.

J. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1.

K. Mean mitochondrial section area in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1. Error bars are SEM from 10 transfectants.

L. Quantification of optineurin staining in J.

Data information: For all graphs, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 4

Figure 4. Lack of Ifnb alters ER/mitochondria platforms and mitochondrial fission.

A. Drp1 oligomerization by chemical crosslinking in wild-type CNs with or without rIFN-β for 2 h.

B. Quantification of A.

C. Immunoblot of INF2 and quantification from cell fractionation of Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 2 h.

D. EM of Ifnb+/+ and Ifnb-/- thalami from 12-month-old mice. Scale bars equal 5 microns. Black arrows indicate mitochondria at a distance < 200 nm, and white arrows indicate distance >200 nm.

E. Volume rendering from 3D TEM images of Ifnb+/+ and Ifnb-/- thalami. Mitochondria are blue, the ER is yellow, and nuclei are grey. Mitochondria undergoing fission are cyan and indicated with a white arrow. 2D pictures are shown in Fig 2.

F. ER/mitochondria closeness, evaluated as the distance from a mitochondrion to the nearest ER structure, extracted from EM images.

G. Contact zone length, quantified as the percentage of the perimeter of a mitochondrion in close contact (<20 nm) with ER, extracted from EM images.

H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci.

I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3.

Data information: For all graphs, error bars mean SEM; * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.001.
IFN-b rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1 (2021)
Figure 5

Figure 5. STAT5 controls Drp1 phosphorylation in neurons by promoting PGAM5 expression.

A. Immunoblot of phospho-S622 and pan-Drp1 in wild-type CNs treated with or without 10 or 50 μM pimozide and with or without 100 U/mL rIFN β. * denotes a potential Drp1 cleavage product of approximatively 50 kDa.

B. Immunoblot of phospho-S622 and pan-Drp1 in N2A cells depleted of STAT5 A and B by CRISPR/Cas9 (DKO) or with nontargeting control (NTC). Vinculin was used as a loading control.

C. Quantification of B.

D. Projections of 3D images from immunostaining for phospho-S622 Drp1 (yellow), Hsp60 (mitochondria, green), and calnexin (ER, magenta) in N2A cells depleted of STAT5 A and B (δStat5) or IFN-β (δIfnb) or with nontargeting control (NTC). Cells with or without rIFN-β treatment for 6 h. Scale bar equals 5 microns. Arrows indicate triple-positive foci.

E. Quantification of D.

F. Pgam5 promotor contains STAT5 binding motifs. Schematic of the upstream region of pgam5, encompassing the pgam5 promotor and pxmp2 with its terminator. The TTCT/CnA/GGAA motif is the optimal STAT5A and B binding motif and is specific to STAT5. OR indicates the origin of replication, as predicted by GPminer. Distances are in bp.

G. Kinetics of the expression of pgam5 by qPCR in CNs on rIFN-β treatment.

H. PGAM5 levels by rtPCR in NTC, δIfnb, and δStat5 N2A cells with and without rIFN-β for 6 h. Error bars are SD from 1 representative graph of 3 independent experiments.

I. Immunoblotting against PGAM5 in Ifnb+/+ ­and Ifnb-/- CNs with or without rIFN-β treatment for 2 h and quantification.

J. Quantification of the impact of rIFN-β on Drp1 phosphorylation after silencing of genes encoding the kinases CDK5, Camk2a, and ERK2 and the phosphorylase PGAM5.

K. Kinetics of the impact of rIFN-β on S622 and S643 phosphorylation. Quantified in J.

L. Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area. Scale bars equal 5 microns. Error bars mean SEM from 10 individual cells.

M. Optineurin immunostaining in Ifnb+/+ and Ifnb-/- MEFs overexpressing PGAM5 and quantification. Empty vector was used as control. Additional controls are shown in Fig EV5L. Error bars are SD from N=30 cells.

N. Oxidative stress in δIfnb N2A cells decreases on overexpression of PGAM5 and phosphomimetic Drp1 but not wild-type Drp1. Immunoblots of PGAM5. Vinculin was used as a loading control.

O. Loss of mitochondrial membrane potential in δIfnb N2A cells. Quantification of TMRE by automated Incucyte-based imaging and analysis. Bars are mean + SEM.

Data information: For all graphs, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.