Fig. 3

Silica and alumina oxide particles synergize with influenza viruses to aggravate lysosomal dysfunction. (A) MTS assay of evaluating the viability of A549 cells treated with PBS, iron oxide (100 μg/mL), silica oxide (100 μg/mL), or alumina (30 μg/mL) combined with vehicle or the indicated amounts of seasonal H1N1 or H5N1 virus 48 h post-infection. (B) Immunoblot analysis of LAMP1 and LAMP2 deglycosylation in A549 cells treated with vehicle, H1N1 (M.O.I., 3) or H5N1 (M.O.I., 0.3) virus combined with PBS, iron oxide (100 μg/mL), silica oxide (100 μg/mL), or alumina (30 μg/mL), separately, at 24 h post-infection. LAMP1 and LAMP2 levels were normalized to β-actin levels. (C) Confocal microscopy analysis of viral NP-positive nuclei in A549 cells treated with vehicle or H1N1 (M.O.I., 3) or H5N1 (M.O.I., 0.3) virus combined with PBS, iron oxide (100 μg/mL), silica oxide (100 μg/mL), or alumina (30 μg/mL) for 4 h. The graph on the right indicates the percentage of NP-positive cells determined using ImageJ software from at least 1,000 cells. Scale bars, 50 μm. (D) q-PCR detection of the influenza virus M1 gene in A549 cells infected with H1N1 (M.O.I., 3) or H5N1 (M.O.I., 0.3) virus combined with PBS, iron oxide (100 μg/mL), silica oxide (100 μg/mL), or alumina (30 μg/mL), separately, at 0.25 h, 0.5 h, 1 h, 2 h, 3 h after infection. The data are presented as the mean ± S.E.M. of three independent experiments. *P < 0.05, **P < 0.01