Additional file 1 of Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile

Additional file 1. 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