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. 2021 Jan 6;12(1):49. doi: 10.1038/s41419-020-03335-7

Fig. 3. Gefitinib elicits mitochondrial damage and subsequent mtROS generation.

Fig. 3

A JC-1 assay to detect loss of mitochondrial membrane potential (MMP) in THP-1 cells. PMA-differentiated THP-1 cells were treated with 20 μM gefitinib or 1 μM Staurosporin for 8 h. MMP was measured using JC-1 probe. Data shown are the mean ± S.D. Significant differences were determined by student’s t-test; ***p < 0.001. B Immunofluorescence imaging of mitochondrial aggregation in gefitinib-treated THP-1 cells. PMA-differentiated THP-1 cells were treated with 20 μM gefitinib for 8 h, and then performed immunofluorescence staining with Tom20 antibody, and 4’,6-diamidino-2-phenylindole (DAPI) nuclear staining (Scale bar, 10 μm). Arrows indicate the aggregated mitochondria. All images are representatives of three independent experiments. C Immunofluorescence imaging of mtROS generation in gefitinib-treated THP-1 cells. PMA-differentiated THP-1 cells were treated with 20 μM gefitinib for 8 h, and then stained with MitoSOX. Fluorescence images and intensity were acquired as described in the materials and methods section. Cell morphology was determined by Nomarski differential interference contrast (DIC) microscopy (Scale bar, 20 μm). All images are representatives of three independent experiments and graphs are shown as the mean ± S.D. Significant differences were determined by student’s t-test; ***p < 0.001. D The inhibitory effect of Mito-TEMPO on gefitinib-induced IL-1β release. PMA-differentiated THP-1 cells were pretreated with 200 μM Mito-TEMPO or 100 μM glyburide for 0.5 h and then treated with 20 μM gefitinib for 8 h. Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. E The inhibitory effect of BHA on gefitinib-induced IL-1β release. PMA-differentiated THP-1 cells were pretreated with the indicated concentrations of BHA for 0.5 h and then treated with 20 μM gefitinib for 8 h. Cell-free supernatants (Sup) and cell lysates were subjected to immunoblotting with the indicated antibodies. F, G ASC oligomerisation assay in THP-1 cells. PMA-differentiated THP-1 cells were pretreated with 200 μM Mito-TEMPO (F) or 100 μM BHA (G) for 0.5 h and then treated with 20 μM gefitinib for 8 h. DSS-mediated crosslinked pellets (Crosslinked-pellet) and soluble lysates (Input) were subjected to immunoblotting with anti-ASC antibody. All data and images in Fig. 3 are representatives of at least three independent experiments.