EMBO/soda-nel huggingface dataset:
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 3

Figure 3. ACM C-MSC show elevated oxidative stress, PPARγ and CD36 expression, and, during adipogenic differentiation, CD36 levels and functionality.

A) Left panel: exemplificative FACS analysis of the FITC emission of unstained HC C-MSC (autofluorescence; white), HC C-MSC in GM treated with DCF (grey), ACM C-MSC in GM treated with DCF (red), and HC cells in GM treated with 2mmol/l H2O2 as positive control (blue). Right panel: mean DCF emission of HC and ACM C-MSC in GM (n=5 biological replicates; Two-tailed Student's t-test).

B) Left panels: representative images of MDA immunostaining (green) on ACM and HC C-MSC in GM. Nuclei are counterstained with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test).

C) GSH/GSSG ratio quantification in HC and ACM C-MSC cultured in GM (n=8 biological replicates).

D) Top panel: representative images of Western Blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARγ antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARγ (n=18 biological replicates) and CD36 (n=17 vs. n=16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test).

E) Results of a FACS analysis of ACM and HC C-MSC, cultured in GM (time-point 0) or AM for 3, 6 or 10 days (time-points 3, 6 and 10, respectively) and marked with anti-CD36 antibody and Nile red. The mean (n=4 biological replicates ACM, n=5 biological replicates HC) fluorescence of CD36 and Nile Red is shown for each condition, the relative regression line, its equation, R2 and p-value (X-Y correlation).

F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova).

Data information: mean ± SEM. * p<0.05, ** p<0.01.
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 4

Figure 4. ACM C-MSC lipogenesis is increased with oxLDL or 13HODE and prevented by NAC treatment.

A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova).

B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova).

C) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova).

Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 5

Figure 5. CD36 silencing reduces lipid accumulation and PPARγ antagonism reduces CD36 expression and function in ACM C-MSC.

A) Left panel: representative images of Western Blot of CD36, PPARγ and GAPDH expression of protein extracts of ACM C-MSC treated with scramble siRNA or CD36 siRNA. Right panels: d.a. normalized on the house-keeping protein GAPDH (n=7 biological replicates; Two-tailed Student's t-test).

B) Left panels: representative images of ORO staining on ACM C-MSC treated with scramble siRNA or CD36 siRNA and cultured in AM with 150µg/ml oxLDL. Right panel: image quantification (n=7 biological replicates; Two-tailed Student's t-test).

C) Left panel: correlation between CD36/GAPDH and PPARγ/GAPDH d.a. Right panel: correlation between CD36/GAPDH d.a. and ORO lipid accumulation. Regression lines, their equations, R2 and correlation p values are shown (X-Y correlation).

D) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC treated with scramble siRNA or CD36 siRNA, cultured in AM and subjected to 10μg/ml DiI oxLDL treatment. Right panel: quantification of the DiI fluorescence normalized on nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test).

E) Left panel: representative images of internalization of oxLDL (red) in ACM C-MSC cultured in AM or AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Right panel: quantification of the DiI fluorescence normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test).

F) Left panel: representative images of Western Blot of CD36 and GAPDH expression of protein extracts of ACM C-MSC cultured in AM or AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH (n=7 biological replicates; Two-tailed Student's t-test).

Data information: mean ± SEM. * p<0.05; ** p<0.01.
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 6

Figure 6. HFD increases plasma oxLDL, cardiac adipose substitution, PPARγ and CD36 expression, and lipid peroxidation, and impairs RV function in ACM mice.

A) Left panel: plasma total cholesterol in the different lipoprotein fractions of WT and Pkp2+/- mice fed a CD (green line) or a 3-month HFD (blue line; n=7). Right panel: plasma total cholesterol quantity in fractions 29-32, corresponding to low, very-low and intermediate-density lipoproteins, of WT and Pkp2+/- mice fed a CD (green line) or a 3-month HFD (blue line; n=7; Mann-Whitney test).

B) Plasma concentration of oxLDL in WT and Pkp2+/- mice samples, fed a CD and a 3-month HFD (n=6; Two-tailed Student's t-test).

C) Left panels: representative images of ORO staining of HFD-fed WT and Pkp2+/- cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3).

D) Representative images of PPARγ (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9).

E) Representative images of MDA (green) immunostaining on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the MDA staining in HFD is shown relative to the values of CD (n=9).

F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Nuclei are counterstained with Hoechst33342 (blue). Quantification of the staining in HFD is shown relative to the values of CD (n=9).

G) RV EF and FS percentages, RVID in systole and diastole of WT and Pkp2+/- mice after (right) 3-month HFD feeding (n=10; Two-tailed Student's t-test).

Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 7

Figure7. Atorvastatin prevented ACM phenotype in HFD-fed Pkp2+/- mice.

A) Left panel: plasma total cholesterol in the different lipoprotein fractions of WT and Pkp2+/- mice fed a 3-month HFD (blue line) or a 3-month HFD+atorvastatin (red line). Right panel: plasma total cholesterol quantity in fractions 29-32, corresponding to low, very-low and intermediate-density lipoproteins, of WT and Pkp2+/- mice fed a 3-month HFD (blue line; n=7) or a 3-month HFD+atorvastatin (red line; n=9; Two-tailed Student's t-test).

B) oxLDL plasma concentration in WT and Pkp2+/- mouse samples, fed a 3-month HFD plus atorvastatin (n=8), compared to HFD (as in Figure 6B; Two-tailed Student's t-test).

C) Left panel: representative images of ORO staining of cardiac sections of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test).

D) Representative images of PPARγ immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test).

E) Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test).

F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD and relative to WT in CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue).

G) RV EF and FS percentages, RVID in systole and diastole of Pkp2+/- mice during (left panel) and after (right panel) 3-month HFD plus atorvastatin feeding (n=9). For comparison RV EF of Pkp2+/- mice fed a HFD is shown (as in Figure 6G; Two-tailed Student's t-test).

Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.
Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 1

Fig. 1. Thioparib is a potent pan-PARP inhibitor and kills BRCA-deficient tumors in vivo.

(A) Structures of thioparib, and its enantiomer Cpd 391.

(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM.

(C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM.

(D) Correlation between cytotoxicity and EC50, as measured by the DSB FA model. The cytotoxicity data are shown in Appendix Table S2. Pearson test was performed to calculate the correlation.

(E) IC50 values of the 13 isoforms of PARPs, as detected by biotinylated NAD+-based luminescence assays. Numbers with superscripts are data from the reference (Antolin et al, 2020).

(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.

Data information: ThP: Thioparib, OP: Olaparib, TP: Talazoparib, RP: Rucapairb, VP: Veliparib, 391: Cpd 391.
Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 2

Fig. 2. Thioparib overcomes multiple PARPi-resistance mechanisms.

(A) Thioparib and olaparib sensitivity of WT and 53BP1#KO (53BP1−/−) MDA-MB-436 cells. Cells were treated with thioparib or olaparib for 7 d and measured by CCK8 assay. Data from three independent experiments are presented as mean ± SEM.

(B) Efficacy of thioparib in the PARPi-resistant CDX model MDA-MB-436 53BP1#KO. Mice bearing tumors (n = 6) were dosed with 10 mg/kg thioparib, 100 mg/kg olaparib, 10 mg/kg simmiparib, or vehicle (Veh) for 3 weeks. Data are depicted as mean ± SEM. ***P < 0.0001, ns: P = 0.7706.

(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.

(D) Efficacy of thioparib in the PARPi-resistant model MDA-MB-436#AZD2281-R1. Nude mice bearing MDA-MB-436#AZD2281-R1 tumors (n = 6) were orally dosed with 40 mg/kg thioparib and 100 mg/kg olaparib for 3 weeks. Mice tails show complete regression of tumors. Data are shown as mean ± SEM. ***P < 0.0001.

(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.

Data information: Statistical analysis was performed by two-way ANOVA. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391, SP: Simmiparib.
Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 3

Fig. 3. Thioparib targets HR-deficient hematologic cancer cells in vitro and in vivo.

(A) The chart indicates alterations of genes involved in the HR pathway in a panel of malignant hematologic cell lines.

(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.

(C) Effects of thioparib on HR-proficient MM1.S (left) and POLQ-deficient MV-4-11 (right) xenografts (n = 4). Mice bearing tumors were dosed orally with 0.3 mg/kg talazoparib, 10 mg/kg thioparib daily (qd), or 30 mg/kg thioparib once every 3 days (q3d). Data shown represent the mean ± SEM. Statistical analysis was performed by two-way ANOVA. Left panel: P = 0.0007, 0.9741, 0.0005; right panel: P = 0.0296, 0.0003, < 0.0001 (top to bottom).

(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.
Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 4

Fig. 4. Thioparib treatment led to PARP1-dependent DNA damage and induced S-phase arrest and apoptosis.

(A) DNA damage as determined by the alkaline comet assay. JeKo-1 and THP-1 cells were treated with the indicated drugs for 12 h before being harvested for the comet assay. Representative photographs of the comet assay are shown; Scale bar: 25 μm.

(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.

(C) and (D) Effects of thioparib on cell cycle and apoptosis. Cell cycle arrest (C) or apoptosis (D) induced by thioparib, talazoparib, and olaparib in JeKo-1 cells were analyzed by PI-staining-based or Annexin V-FITC-based flow cytometry. Data from three independent experiments are expressed as the mean ± SD. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001, *P = 0.0131.

(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.

(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.
Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 6

Fig. 6. Thioparib induces a type I interferon (IFN) response in tumor cells through PARP7 inhibition in vitro and in vivo.

(A) IFN-β mRNA levels in JeKo-1 cells treated with the indicated drugs for 12 h. Data from three biological replicates are depicted as mean ± SD. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001, **P = 0.0013.

(B) Concentration-dependent increase in p-STAT1 by thioparib in JeKo-1 and THP-1 cells.

(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).

(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.

(E) The changes in IFNB1 and CXCL10 mRNA levels in HT-29 parent, PARP1, and PARP7 KO cells exposed to thioparib. Data from three biological replicates are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Upper panel: the absolute mRNA levels by normalizing against the parent untreated group, and P value was calculated compared to the parent untreated group, *P = 0.0125, 0.0105, 0.0187, 0.0349 (from left to right), **P = 0.0021, ***P < 0.0001. Lower panel: the relative mRNA fold change normalized against the untreated group in each cell line, and P value was calculated compared to the untreated group in each cell line, ns: P = 0.3194, 0.3373, 0.4158, 0.4907, **P = 0.0070, ***P < 0.0001, = 0.0009, < 0.0001, <0.0001, <0.0001, <0.0001, <0.0001 (from left to right).

(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.

(G) Antitumor activity of 10 mg/kg thioparib in MC38 syngeneic tumor model. Tumor-bearing C57BL/6J mice were dosed orally with vehicle or thioparib for 21 days (n=6). P value was determined by two-way ANOVA, ***P < 0.0001. QD, once daily. Data are presented as the mean ± SEM.

(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.

(I) Antitumor activity of 10 mg/kg thioparib in C57BL/6J subcutaneous tumor model using MC38 PARP1 KO or PARP7 KO cells (n = 6). P value was determined by two-way ANOVA. ***P < 0.0001, ns: P = 0.0597. Data are presented as the mean ± SEM.
Polarized Cortical Tension drives Zebrafish Epiboly Movements (2016)
Figure 1

Figure 1. Epiboly kinematics

(a) Representation of a zebrafish embryo at 70 % epiboly. Animal (A) and vegetal (V) poles and φ (embryo surface - red) and  (internal to external axis - green) are indicated. The enveloping layer (EVL) is a single-cell layer (dark grey) that stands at the animal cap surface surrounding the inner deep cells (DCs) (blue). It is attached to the yolk cell membrane (purple). The YSL and Yolk Cytoplasmic Layer (YCL) (yellow) at the periphery and yolk granules fill the yolk cell. Progression of epiboly is represented by a curved black arrow. Geometrical axes are shown in grey. (b) Kinematics of the embryo surface. A kymograph of the embryo surface (φ axis) from a Tg (β-actin:m-GFP) embryo shows the continuous displacement of the EVL / yolk margin, which accelerates after 50% epiboly, while the yolk cell membrane does not show any shift (see Movie EV1). (c) Theoretical kymograph from 50 % to 70 % epiboly superimposed, between the red lines, on the experimental kymograph (b). Membrane signal intensity is color-coded (blue to red). White arrowheads point to flow lines (black). (d) AV velocities, normalized to EVL / yolk margin velocity, were measured by PIV of time-lapse recordings of the surface of lectin-soaked embryos. The plots correspond to three different epiboly stages (30 %, blue; 60 %, red; and 90 %, green). Blue and red arrows point to the width of the E-YSL at 30 and 60 % epiboly respectively (from Appendix Figure S1). (e) The yolk cell membrane is highly convoluted (white brackets) ahead of the EVL leading cells and condenses over time. Tg (β-actin:m-GFP) embryo at 30 % (left) and 60 % (right) epiboly. Scale bar 25 μm. Inset shows a high magnification of the wrinkled area (arrowhead). Scale bar 10 μm. (f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.
Polarized Cortical Tension drives Zebrafish Epiboly Movements (2016)
Figure 2

Figure 2. Yolk Flows

(a) Yolk granules flow in stereotyped patterns as epiboly progresses. PIV of time-lapse snapshots imaged by two-photon microscopy of a Tg (β-actin:m-GFP) embryo (from Movie EV2). Upon epiboly onset, yolk granules flow towards the animal pole displaying two internal vortices near the E-YSL. At 50 %, the movement of the yolk granules halts, but resumes afterwards until the end of epiboly. Animal (A) and vegetal (V) poles are indicated. Scale bar 100 μm. (b) Internal yolk content speed (μm/min) at the center of the embryo, over time (min). Inset: Spherical coordinates. The main axis of the sphere is represented by z, from the animal to the vegetal pole. The polar angle is represented by φ and the radial coordinate is denoted . (c) Vortex midpoints distance from the center (), over time (min). (d) Vortex midpoints position in relation to the φ axis, over time (min). Black lines represent individual measurements. Red lines represent averages. (e) Correlation between vortex displacement (blue, average of n=3 embryos) vs. EVL margin displacement (red, average of n = 4 embryos) along the main axis (z), over time (min). Standard Errors are displayed. Arrow points to 50 % epiboly.