Fig. 4
CD-NPs induced acute mitochondrial damage in vitro and in vivo. A The mitochondrial damage illustrated by TEM after treatment with 300 µg/mL mixed CD-MPs and CD-NPs for 24 h in vitro and with 32 μg mixed CD/day for 2 weeks in vivo. Green arrows point to endocytosed CD particles. Red arrows point to damaged mitochondria stimulated by CD particles. B Mitochondrial membrane potential was detected by JC-1 staining after treatment with 300 µg/mL mixed CD-MPs and CD-NPs for 24 h. Data were expressed as the mean ± SD, n = 3. ***P < 0.001. C The content of intracellular free Ca2+ was detected by indirect immunofluorescence after treatment with 300 µg/mL mixed CD-MPs for 8 h and 300 µg/mL mixed CD-NPs for 8 and 12 h. Data are expressed as the mean ± SD, n = 3. **P < 0.01 and ***P < 0.001. n.s., no significance. D The ROS level of cells was detected by indirect immunofluorescence after treatment with 300 µg/mL mixed CD-MPs for 8 h or 300 µg/mL mixed CD-NPs for 8 and 12 h. Data were expressed as the mean ± SD, n = 3. ***P < 0.001. E The level of HO-1 protein was detected by western blot after treatment with 300 µg/mL mixed CD-MPs for 8 h and 300 µg/mL mixed CD-NPs for 8 and 12 h. Data were expressed as the mean ± SD, n = 3. **P < 0.01 and ***P < 0.001. n.s. no significance, CD-MPs coal dust micron particles, CD-NPs coal dust nanoparticles, CD coal dust, TEM transmission electron microscope, ROS reactive oxygen species. Remarks: Due to insufficient experimental funds, in the western blot assay, we cut the PVDF membrane into a membrane small enough so the antibody could be incubated according to its molecular weight and the protein molecular weight marker