Additional file 1 of Use of an in silico knowledge discovery approach to determine mechanistic studies of silver nanoparticles-induced toxicity from in vitro to in vivo

Additional file 1: Fig. S1. Physicochemical features of the four types of synthetic AgNPs (SCS, LCS, SAS and LAS). (A) Particle size and morphology, revealed by transmission electron microscopy (TEM) (Scale bar: 20 nm, for SCS and SAS; 100 nm, for LCS and LAS); (B) Chemical composition, analyzed by energy-dispersive X-ray (EDX) spectrometry; (C) and (D) Particle size distribution (in water- and medium-based suspensions, each), measured by the dynamic light scattering (DLS) method; (E) Absorbance spectrum (in water- or medium-based suspensions), determined by UV–Vis spectrophotometry. Fig. S2 Single-cell-line-versus-four-AgNP-types dose–response patterns at 24 and 48 h post-exposure. (A) BEAS-2B; (B) Clone 9; (C) HaCaT; (D) HEK293; (E) THP-1; (F) IEC-6; and (G) AML12, which are reorganized from the cell viability results depicted in Figs. 1 and 2. *(P < 0.05), **(P < 0.01) and *** (P < 0.005) denote significant differences in cell viability between different particle type groups. Fig. S3 The influence of exposure to SCS, LCS, SAS, or LAS on the statuses of cell death modalities (apoptosis, necrosis, and autophagy) occurring in IEC-6 cells. (A) and (B) Apoptotic and necrotic events in response to SAS exposure (1, 5, 10 and 15 µg/ml) for 24 h, as measured by flow cytometry using annexin V-FITC/propidium iodide (PI) staining (Annexin V-FITC positive/PI negative cells are those undergoing early-stage apoptosis, while annexin V-FITC positive/PI negative cells are those in the late stage of apoptosis. Necrotic cells are considered to be stained with PI alone. Total apoptotic cells = early apoptotic cells + late apoptotic cells); (C) Autophagic events in response to respective exposures to SCS, LCS, SAS, and LAS (5 µg/ml) for 8 h, as measured by flow cytometry with acridine orange (AO) staining. (D) Time-course analyses of the autophagic activity in response to serial doses of SAS (0.5, 1, 5, 10 and 15 µg/ml). Results were representative of three independent experiments performed in triplicate. *(P < 0.05), **(P < 0.01) and *** (P < 0.005) denote significant differences between the control and treatment groups. Fig. S4 Cellular senescence and proliferative inhibition evoked by longer exposures to noncytotoxic doses of LCS particles. (A) Different degrees of senescence-associated β-galactosidase (SA-β-gal) activity in untreated control and LCS-treated AML12 cells (1, 5 and 10 µg/ml; 48 h post-treatment); (B) The capacity of AML12 cells to survive and proliferate following challenge with LCS (1 and 5 µg/ml) for 48 h, as evaluated by the clonogenic assay. (C) Quantification of the colony-forming capacity of untreated control and LCS-treated cells (indicated by “surviving fraction (SF)”; please refer to “clonogenic assay” in the following supplementary materials and methods). *(P < 0.05), **(P < 0.01) and *** (P < 0.005) denote significant differences between the control and treatment groups.