Figure S1. Structural and morphological characterization of USGO and LGO. A) and B) Height AFM images, C) and D) scanning electron micrographs. Figure S2. Lung inflammation after single exposure to GOs. A) Immune cells infiltration and granulomatous formation in mice lungs at 1, 7 and 28 days after exposure to a single dose of carbon nanomaterials (USGO, LGO or MWCNT) compared to water for injection (= control). Lung infiltrates (Red arrow), granulomas formation (green arrows). Figure S3. DNA damages in lung cells after single exposure. (A) Quantification of γ-H2Ax positive nuclei of epithelial cells (E-Cadherin+) in lung parenchyma (outside inflammatory infiltrates and excluding CD45+ cells). (B) Quantification of immune cells in mice lungs (number of CD45+ cells per FOV, n = 7) with immunostaining using Alexa Fluor® 594 labelled rat anti-mouse CD45 monoclonal antibody. A single exposure to high dose of carbon nanomaterials (USGO, LGO or MWCNTs) compared to water for injection (= control). Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 3). Figure S4. DNA damages in lung epithelial cells after repeated exposure. Quantification of γ-H2Ax positive nuclei of epithelial cells (E-Cadherin+) in lung parenchyma (outside inflammatory infiltrates and excluding CD45+ cells) was performed after repeated exposure (x3) to high dose (A) or low dose (B) of carbon nanomaterials (USGO, LGO or MWCNTs), compared to water for injection (= control). Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 3). Figure S5. DNA repair activity in whole lung tissue after repeated exposure to a high dose. (A - C) mRNA expression level measured by RT-qPCR for three genes coding for DNA repair proteins, expressed as fold change, in whole lung tissue lysate after exposure to carbon materials (USGO, LGO or MWCNTs) or negative control (water for injection). Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 6). Figure S6. Published data on inflammation markers after repeated exposure to a high dose. (A - C) BALF cell counts measured after repeated exposure of mice to carbon materials (USGO, LGO or MWCNTs) or negative control (water for injection). (D) mRNA expression level measured by RT-qPCR for SAA3, expressed as fold change, in whole lung tissue lysate after exposure to carbon materials (USGO, LGO or MWCNTs) or negative control (water for injection). (E-H) Pro-inflammatory cytokines measured by ELISA multiplex. Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 6). From [ref 26; Loret et al, Advanced Science, 2022;9:2104559] Figure S7. Published data on oxidative stress markers after repeated exposure to a high dose. (A - E) mRNA expression levels measured by RT-qPCR for different genes involved in the regulation of oxidative stress, expressed as fold change, in whole lung tissue lysate after exposure to carbon materials (USGO, LGO or MWCNTs) or negative control (water for injection). Statistical significance: *P<0.05, **P<0.01, ***P<0.001. (n = 6). From [ref 26; Loret et al, Advanced Science, 2022;9:2104559]. Figure S8. Evaluation of DNA damages after repeated exposure to a high dose of GO sheets or MWCNTs. Lung parenchyma, infiltrates, granulomatous areas and nanomaterial deposition (brown colour) are highlighted in the H&E staining. The corresponding DNA damages after immunostaining (IHC) with γ-H2AX (DAB+ nuclei) and counterstaining with haematoxylin are highlighted. The framed inset pictures represent the same area of IHC without incubation with the primary antibody. The scale bar is equivalent to 20 µm. Table S1. Summary of the physicochemical characterization of USGO and LGO sheets. Table S2. List of PCR primers. Table S3. Pearson R values (n = 6) for USGO, day 1. Table S4. P values (n = 6) for USGO, day 1. Table S5. Pearson R values (n = 6) for USGO, day 7. Table S6. P values (n = 6) for USGO, day 7. Table S7. Pearson R values (n = 6) for USGO, day 28. Table S8. P values (n = 6) for USGO, day 28. Table S9. Pearson R values (n = 6) for USGO, day 84. Table S10. P values (n = 6) for USGO, day 84. Table S11. Pearson R values (n = 6) for LGO, day 1. Table S12. P values (n = 6) for LGO, day 1. Table S13. Pearson R values (n = 6) for LGO, day 7. Table S14. P values (n = 6) for LGO, day 7. Table S15. Pearson R values (n = 6) for LGO, day 28. Table S16. P values (n = 6) for LGO, day 28. Table S17. Pearson R values (n = 6) for LGO, day 84. Table S18. P values (n = 6) for LGO, day 84. Table S19. Pearson R values (n = 6) for MWCNT, day 1. Table S20. P values (n = 6) for MWCNT, day 1. Table S21. Pearson R values (n = 6) for MWCNT, day 7. Table S22. P values (n = 6) for MWCNT, day 7. Table S23. Pearson R values (n = 6) for MWCNT, day 28. Table S24. P values (n = 6) for MWCNT, day 28. Table S25. Pearson R values (n = 6) for MWCNT, day 84. Table S26. P values (n = 6) for MWCNT, day 84.

H2020 Future and Emerging Technologies UK Research and Innovation

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