Fig 2: Representative images of immunostaining for DNA damages in lung sections after repeated exposure to a high dose of materials. DNA damages was performed using rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139, and donkey anti-rabbit Alexa Fluor 647. Mice were exposed by single oro-pharyngeal aspiration to a high dose GO sheets (USGO or LGO), MWCNTs, or water for injection. All sections were counterstained for DAPI. Scale bar = 50 µm. Inset boxes highlight positive nuclei with higher magnification (100×)

Fig 3: Experimental design of the study. Single (high-dose) and repeated (low and high-dose) exposure to nanometric (USGO) or micrometric (LGO) graphene oxide sheets or MWCNTs were delivered to mouse lungs by oro-pharyngeal aspiration. Quantification of DNA damages in formalin-fixed paraffin embedded lung sections was performed using recombinant rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139, combined with anti-mouse CD45 Alexa Fluor 594 and anti-mouse E-Cadherin Alexa Fluor 488 to phenotype the damaged cells (n = 3). A correlation matrix was performed for the repeated exposure (high-dose) study using inflammation parameters obtained from BALF, whole lung ELISA, and RT-qPCR obtained from the same animals (n = 6). Figure created with BioRender.com

Fig 4: Evaluation of DNA damages induced by GO sheets after repeated exposure. A Quantification of lung DNA damages using rabbit anti-mouse γ-H2Ax-Phosphorilated Ser139 immunostaining after repeated exposure to 3 × 10 µg (high dose) of GO sheets (USGO or LGO) and MWCNTs (positive control/reference materials), expressed as fold change in fluorescence intensity (negative control = water for injection). B Percentage of γ-H2Ax imunoreactivity (fluorescence intensity) located in lung immune cell infiltrates or in lung parenchyma at day 1, 7, 28 and 84 after repeated exposure to 3 × 10 µg. C Quantification of lung DNA damages after repeated exposure to 3 × 1 µg (low dose) of materials. D Percentage of γ-H2Ax imunoreactivity (fluorescence intensity) located in lung immune cell infiltrates or in lung parenchyma at day 1, 7 and 28 after repeated exposure to 3 × 1 µg. E Total number of γ-H2Ax positive cells in lung parenchyma (outside inflammatory infiltrates) expressed as fold change in fluorescence intensity, after repeated exposure to 3 × 10 µg. F Percentage of E-cadherin+ γ-H2Ax+ cells and CD45+ γ-H2Ax+ cells in lung parenchyma at day 1, 7, 28 and 84 after exposure. G Total number of γ-H2Ax positive cells in lung parenchyma (outside inflammatory infiltrates) expressed as fold change in fluorescence intensity, after repeated exposure to 3 × 1 µg. H Percentage of E-cadherin+ γ-H2Ax+ cells and CD45+ γ-H2Ax+ cells in lung parenchyma at day 1, 7 and 28 after exposure. Significance level *p < 0.05 **p < 0.01, ***p < 0.001 (One-Way ANOVA; n = 3)

Fig 5: PARP1/PAR pathway is activated in overexpressed α‐synucleinA53T models of Parkinson's disease. (a) SN4741 cells were transfected with Adenovirus‐Ctrl/A53T for 36 hr. Representative immunoblots and quantification of the levels of PAR, PARP1, and γ‐H2A.X. Means ± SEM, n = 3. (b) Representative immunoblots and quantification of the levels of PAR, PARP1 and γ‐H2A.X accumulation in SNpc and Cortex section of wild‐type (WT) or α‐synucleinA53T‐tg mice. Means ± SEM, n = 3. (c) Immunohistochemistry (IHC) staining of PARP1 in SNpc and Cortex tissue of WT and α‐synucleinA53T‐tg mice. Statistical analysis of the scores of PARP1 staining also shown in c. Scale bars, 100 μm. (d,e) NAD+ (d) and ATP (e) levels inSN4741 cells measured by spectrophotometer. (f) Representative images of mitochondrial tracker (red) staining from SN4741 cells transfected with Adenovirus‐Ctrl/A53T, and quantification of mean branch length of Mitochondrial fragments. Means ± SEM, n = 20. Scale bars, 5 μm. (g) Representative images of TMRM (red) staining and quantification of fluorescence intensity from SN4741 cells. Means ± SEM, n = 20. Scale bars, 15 μm. (h) Apoptosis rates were measured by fluorescence‐activated cell sorting analysis. n = 3 (the statistical significantly was analyzed by unpaired Student's t test, *p < .05, **p < .01 and ***p < .001). PARP1, Poly (ADP‐ribose) polymerase 1; TMRM, tetramethylrhodamine methyl ester