Figure 3

DRP1 is required for mitochondrial fragmentation upon A. baumannii infection. (A) The illustration represents the canonical mitochondrial fission pathway (created with BioRender.com). (B) A549 cells were infected with the indicated bacteria for 6 h at MOI 50. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and anti-DRP1 antibody (green). Scale bar represents 10 µm. (C) Western blot on A549 cell lysates after indicated treatments. Anti-DRP1 antibody was used to examine DRP1 levels and ß-actin served as the loading control. (D) A549 cells treated with DRP1 siRNA or control (scrambled) siRNA were infected with wildtype A. baumannii (Ab19606) for 6 h at MOI 50. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red), anti-DRP1 antibody (green) and DAPI to label the nucleus (blue). Scale bar represents 10 µm. (E) The scatter plots represent the quantification of mitochondrial area (red) and perimeter (orange). The experiment was performed in triplicates, n = 75–132 cells. Error bars represent standard deviation. One-way ANOVA with Tukey’s multiple comparisons test **p \(\le \) 0.01, ****p \(\le \) 0.0001 (F) A549 cells pre-treated with Mdivi1 (10 µM) or DMSO control were infected with wildtype A. baumannii (Ab19606) for 6 h at MOI 50. Immunofluorescence was performed using anti-TOM20 antibody to stain mitochondria (red) and DAPI to label the nucleus (blue). (G) The scatter plots represent the quantification of mitochondrial area (red) and perimeter (orange). The experiment was performed in triplicates, n = 88–138 cells. Error bars represent standard deviation. One-way ANOVA with Tukey’s multiple comparisons test ****p \(\le \) 0.0001. (H) Cytotoxicity was assessed by LDH release assay after 6 h of infection in A549 cells treated with the indicated siRNA and bacteria. The experiments were done in triplicates. Error bars represent standard deviation. Two-tailed p value using unpaired t-test ****p \(\le \) 0.0001. % cytotoxicity was calculated by normalizing the LDH release in the infected groups with uninfected/untreated cells (representing no cytotoxicity) and TritonX-100 treated cells (representing the highest cytotoxicity). Mitochondrial area and perimeter quantifications were performed using an unbiased automated CellProfiler pipeline (see “Materials and methods” section for details). All the experiments shown here were performed three times independently.