EMBO/soda-nel huggingface dataset:
Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth (2022)
Figure 6

Fig. 6: PSC serves as a source of dopamine during lymph gland development .

(A-I'') Representative lymph gland images at 48, 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland.

(A-C'') The control profile of lymph gland and PSC iDopamine levels at 48h (A-A''), 72h (B-B'') and 96h (C-C'').

(D-I'') Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h (D-D'' and G-G''), 72h AEL (E-E'' and H-H'') and at 96h AEL (F-F'' and I-I'').

(J) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=33, Antp-Gal4; UAS-GFP; THRNAi, n=8, Antp-Gal4; UAS-GFP; DdcRNAi, n=14), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=26, Antp-Gal4; UAS-GFP; THRNAi, n=25, Antp-Gal4; UAS-GFP; DdcRNAi, n=20) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17).

(K) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=28, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=18), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=25, Antp-Gal4; UAS-GFP; THRNAi, n=24, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=16, Antp-Gal4; UAS-GFP; DdcRNAi, n=18).

(L) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL (Antp-Gal4; UAS-GFP/+, n=19, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=17, Antp-Gal4; UAS-GFP; THRNAi, n=18, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=14, Antp-Gal4; UAS-GFP; DdcRNAi, n=15)

Data information: DNA is stained with Hoechst in blue, Antp-Gal4; UAS-GFP (representative of Posterior Signaling Center, PSC) in green, iDopamine in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in J, K and L represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in J, K and L is Two-way Anova with Tukey's multiple comparisons test. The control animals Antp-Gal4; UAS-GFP/+ were analysed by One-way Anova with Tukey's multiple comparisons test across time points. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line.
Chemogenetic profiling reveals PP2A-independent cytotoxicity of the proposed PP2A activators iHAP1 and DT-061 (2022)
Figure 2

Figure 2. DT-061 and iHAP1 toxicity is not affected by knockdown of specific B56 subunits.

A-C) A stable inducible HEK-293T cell line expressing myc-PP2AC (panel A and B) and a stable inducible HeLa cell line expressing myc- PP2R1A (C) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC and myc-PP2R1A affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting (A and C) and myc-PP2AC by quantitative mass spectrometry (B). A) representative of 3 independent experiments and C) representative of 2 technical repeats and B) analysis of a biological triplicate.

D) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.

E) Doxycyclin-inducible CRISPR-Cas9 HeLa cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of DT-061 or iHAP1 measured after 12 days of treatment. Mean and SD of three independent experiments are shown.
Chemogenetic profiling reveals PP2A-independent cytotoxicity of the proposed PP2A activators iHAP1 and DT-061 (2022)
Figure 4

Figure 4. iHAP1 is a microtubule poison.

A) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics. Taxol and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.

B) Representative colour-coded temporal projection of U2OS EB1-GFP cells following indicated treatment. Scale bar 10µm.

C) Quantification of microtubule dynamics acquired from manual tracking of EB1-GFP comets as indicators for MT growth after drug treatment. The mean and SD are plotted from three independent experiments (n = 10 cells per experiment).

D) Representative temporal projection of mitotic spindles of U2OS EB1-GFP cells following specified treatment. DNA counterstained with SiR-DNA shown in cyan in merged images. Scale bar 10µm.

E) Analysis of number of EB1-GFP comets per mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C in).

Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). See methods for more details. ns, not significant, ****p<0.0001.
Machine Learning Identifies Molecular Regulators and Inhibitors for COVID19-induced Cytokine Release (2021)
Figure 1

Figure 1. SARS-Cov-2 Spike subunit S1 protein causes a significant increase in the expression and release of a panel of cytokines in THP1 cells and human PBMCs.

A. A schematic showing major SARS-CoV-2 proteins and domain structure of spike protein.

B. Changes in the expression of cytokines in THP1 macrophages (left) and PBMCs (right) upon treatment with full-length S1 subunit at 1 µg/ml for 24 hr.

C. Changes in the expression of cytokines in THP1 macrophages upon treatment with different domains of S1 subunit at 1 µg/ml for 24 hr.

D. Measurement of cytokine release from healthy donor PBMCs treated with PBS or NTD at 1 µg/ml for 24 hr.

E. Effect of an anti-RBD antibody on S1 subunit stimulated changes in the expression of cytokines in PBMCs. Left, a schematic showing the domain structure of S1 spike protein and anti-RBD antibody. Right, changes in cytokine gene expression in response to S1 spike protein in the presence or abscene of anti-RBD antibody. Gene expression was measured by qPCR. Cytokine release in the conditioned media was measured by Luminex. Full-length S1 and S1 subunits are purified from HEK293 cells.

Data information, in (B, C,E), data are shown as the mean of three technical replicates; in (D), data are shown as the mean of 20 individual donors. Error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Multiple t-test.
Machine Learning Identifies Molecular Regulators and Inhibitors for COVID19-induced Cytokine Release (2021)
Figure 5

Figure 5. Ponatinib alleviates symptoms of acute lung inflammation in LPS-induced lung inflammation mouse model.

A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages.

B. Ponatinib inhibits LPS-mediated cytokine release in THP1 macrophages in vitro.

C. A schematic showing the overall design of in vivo study evaluating the efficacy of ponatinib in LPS-induced lung inflammation mouse model.

D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).

E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex.

Data information, in (A-B), data are shown as the mean of three technical replicates. Error bars denote SEM. In (E), data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.

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IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 1

Figure 1. Pertussis toxin induces IL-1β expressing myeloid cells after MOG/CFA immunization dependent on IL-1R1 signaling.

A. Frequencies and total numbers (mean +SEM) of CD11b+ myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice.

B. Total numbers (mean +SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h.

C. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β, isolated from LN and restimulated with GM-CSF shown in (B).

D. Frequencies and total numbers (mean +SEM) of CD11b+ myeloid cells isolated from the spleen of immunized mice.

E. Analysis of IL-1R1 expression by CD11b+ cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD-CD11b+ cells with mean frequencies per group.

F. Quantification (mean +SEM) of IL-1β expression by CD11b+ cells shown in (E).

G. Analysis of IL-1β expressing CD11b+ cells shown in (E). Data are representative FACS plots, gated on VD- CD11b+IL-1β+ cells with mean frequencies per group.

H. Quantification (mean +SEM) of myeloid cell populations expressing IL-1β shown in (G).

Neutrophils were defined as CD11b+Ly6G+Ly6Clow cells, monocytes/macrophages as CD11b+Ly6G-Ly6Chigh cells. Cells were isolated (A-C) from naïve non-immunized mice n=5 of each genotype; (D-H) at day 7 after immunization from n=4 WT PBS, n=3 IL-1R1-/- PBS, n=4 WT PTx, n=3 IL-1R1-/- PTx MOG/CFA immunized mice. Cells (A, C, D-H) were restimulated with 20 ng/ml GM-CSF and (B) with GM-CSF or 500 ng/ml LPS in the presence of Monensin for 4 h. *p < 0.05, **p < 0.01, N.S. – not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 2

Figure 2. IL-1R1 expression by murine and human Th17 cells.

A-D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization; and (C) CD4 T cells and (D) Th17 cells isolated from the CNS at the peak of EAE.

Cells (A-D) were restimulated in the presence of MOG for 6 h. Data consist of n=4 wild type mice immunized with MOG/CFA/PTx.

E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.

Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.

Data (A, C) are representative FACS histogram overlay of VD-TCRβ+CD4+ cells gated on IL-17A+, IL-17A-IFNγ+ (IFNγ+), IL-17A-GM-CSF+ (GM-CSF+) or IL-17A-FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean +SEM). Data (E) are FACS plots of cells gated on CD14-CD3+CD4+ cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 3

Figure 3. GM-CSF expressing Th17 cells are expanded by PTx in response to IL-1 signaling.

A. Analysis of cytokine expression by CD4 T cells isolated from the spleen. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+ cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group.

B. Frequencies and total numbers (mean +SEM) of MOG-specific Th17 cells isolated from the dLN.

C-D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D).

Cells (A-D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n=5 WT PBS, n=4 WT PTx, n=4 IL-1R1-/- PBS, n=4 IL-1R1-/- PTx, MOG/CFA immunized mice. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. – not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 4

Figure 4. T cell specific deletion of IL-1R1 impairs Th17 cell expansion.

A. Analysis of CD4 T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+ cells with mean frequencies per group.

B. Analysis of cytokine expression by CD4 T cells isolated from the spleen and restimulated with MOG for 6 h. Data are representative FACS plots, gated on VD-TCRβ+CD4+CD44+ cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group.

C. Total cell numbers (mean +SEM) of MOG-specific Th17 cells isolated from the spleen, shown in (B, upper row).

D. Frequencies and total numbers (mean +SEM) of MOG-specific GM-CSF+IFNγ+/- Th17 cells isolated from the spleen, shown in (B, lower row).

Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. – not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 5

Figure 5. Mice with T cell specific deletion of IL-1R1 are only partially resistant to EAE induction.

A-C. (A) Mean, (B) maximal EAE clinical scores and (C) area under the curve (AUC) of mice immunized with MOG/CFA/PTx. Data shown as (A) mean (+SEM), (B, C) individual plots with mean and are combined from at least four independent experiments and consist of n=41 WT, n=24 IL-1R1ΔT, n=18 IL-1R1-/- mice. *p < 0.05, ***p < 0.001; two-tailed unpaired t-test. Black (*) signs in (A) represent statistical analysis of IL-1R1ΔT vs WT and blue (*) of IL-1R1ΔT vs IL-1R1-/- groups.

D-E. (D) Maximal EAE clinical scores (individual plots with mean) of selected mice and (E) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.

F-H. Analysis of cytokine expression by CD4 T cells isolated from the CNS of mice shown in (D). Data (F) are representative FACS plots, gated on VD-CD90+CD4+CD44+ cells (upper row) with mean frequencies among CD4 T cells per group ± SEM, and (lower row) with mean frequencies per group. (G) Mean fluorescent intensity (mean +SEM) of IL-17A staining by MOG-specific Th17 cells, shown in (F, upper row). (H) Quantification of MOG-specific GM-CSF+ Th17 cells (mean +SEM) shown in (F, lower row).

I-K. Analysis of Treg cells isolated from mice shown in (D). (I) Total Treg cell numbers (mean +SEM) isolated from CNS and dLN. (J) Ratio of Treg and Th17 cells isolated from the CNS. Data are representative FACS plots, gated on VD-CD90+CD4+ cells with mean frequencies per group. (K) Quantification of Treg/Th17 cell ratio (mean +SEM) within CNS and dLN.

L-N. Analysis of CCR6 expression by CD4 T isolated from the dLN and CNS of EAE diseased mice. Data (L) are representative FACS histogram overlays of (black) control, (blue) IL-1R1ΔT cells, gated on CD3+CD4+IL-17A+ cells; and of (black filled) control, (blue filled) IL-1R1ΔT cells, gated on CD3+CD4+IL-17A- cells. (M) Mean fluorescent intensity (mean +SEM) of CCR6 staining by CD4 T cells, shown in (L). (N) Mean fluorescent intensity (mean +SEM) of CCR6 staining by GM-CSF+ and GM-CSF- Th17 cells isolated from CNS, shown in (L).

Cells (F-H) were restimulated with MOG for 6 h and (I-N) in the presence of PMA/Ionomycin for 4 h. Data (D-K) consist of n=4 WT, n=3 IL-1R1ΔT, n=3 IL-1R1-/- mice and (L-N) of n=5 mice of each genotype. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. – not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 6

Figure 6. IL-23 increases numbers of encephalitogenic IL-1R1 deficient Th17 cells.

A-C. Analysis of (A) IL-17A expression by wild type CD4 T cells, followed with the analysis of (B) GM-CSF and (C) GM-CSF and IFNγ co-expression by Th17 cells.

D-G. Analysis of cytokine expression by cells isolated from WT (black), IL-1R1ΔT (blue) and IL-1R1-/- (red) mice and cultured with or without IL-23. Data (D) are representative FACS plots, gated on VD-TCRβ+ cells with mean frequencies per group. Quantification (mean +SEM) of (E) MOG-specific Th17 cells, (F) GM-CSF+ Th17 cells and (G) GM-CSF+IFNγ+ Th17 cells.

Cells (A-G) were isolated from dLN and the spleen of MOG/CFA immunized mice, cultured under indicated conditions for four days and restimulated with MOG for 6 h prior to analysis. Data consist of n=4 of each genotype.

H-I. (H) Mean EAE clinical scores (+SEM) of Rag1-/- mice which received 1 x 105 IL-17A+ cells of indicated genotypes and (I) total cell numbers (mean +SEM) of infiltrates isolated from CNS and used for further analysis.

J-L. Analysis of cytokine expression by CD4 T cells isolated from CNS of mice shown in (H) and restimulated with MOG for 6 h. (C) Data are representative FACS plots, gated on VD-TCRβ+ cells (upper row) with mean frequencies ± SEM. (D) Total cell numbers (mean +SEM) of Th17 cells shown in (C, upper row). (F) Frequencies and total numbers (mean +SEM) of GM-CSF+IFNγ+/- Th17 cells shown in (C, lower row).

Data (H-L) consist of Rag1-/- mice which received WT (n=6), IL-1R1ΔT (n=3) and IL-1R1-/- (n=4) cells polarized in the presence of IL-23 prior to transfer. All experiments were performed at least twice with similar results.
IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells (2016)
Figure 7

Figure 7. Treg depletion results in EAE susceptibility of IL-1R1 deficient mice.

A. Mean EAE clinical scores (+SEM).

B. Maximal EAE clinical scores (individual plots with mean) of selected mice.

C. Total cell numbers (mean +SEM) of infiltrates isolated from the CNS of mice, shown in (B).

D. Analysis of IL-1R1 expression by Th17 isolated from CNS of EAE diseased mice, shown in (B). Data are representative FACS histogram overlays of cells isolated from (grey) wild type mice treated with PBS, (black) wild type, (blue) IL-1R1ΔT and (red) IL-1R1-/- mice treated with PC61, gated on VD-TCRβ+CD4+IL-17A+ cells.

(E) Frequencies of Th17 cells positive for IL-1R1, shown in (D).

Cells (C, D, E) were restimulated in the presence of MOG for 6 h. Data consist of (A) n=10 WT, n=4 IL-1R1-/- PBS, n = 4 WT, n=4 IL-1R1ΔT and n=5 IL-1R1-/- PC61 treated mice and of (B-E) n=4 WT PBS, n = 4 WT, n=3 IL-1R1ΔT and n=3 IL-1R1-/- PC61 treated mice. **p < 0.01, ***p < 0.001, N.S. – not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 1

Figure1. Loss of EWS regulates expression of DDR proteins and causes PARP1 activation

(A) Schematic of SILAC-mass spectrometry. Wild-type (WT) and Ews-/- mBA cells were labeled by heavy or light amino acid, respectively. Differently labelled proteins were analyzed by mass spectrometry. (B) Network analysis of mass spectrometry results. (C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01. (D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert). (E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 2

Figure 2. Loss of EWS induces accumulation of PARP1 at damaged DNA sites.

(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot. (C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm. (D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm. (E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 3

Figure 3. Loss of EWS induces PARP1-dependent PARylation.

(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours). (B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage (C) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm. (D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage. (F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.