Autophagy contributes to bacterial killing by mast cells <i>in vitro</i> following <i>P. aeruginosa</i> infection.

<p>HMC-1 5C6 cells stably expressing non-targeting (NT) shRNA or shRNA specific for the autophagy genes Atg5 or Atg7 were subjected to western blot analysis to confirm protein knockdown (A). Numbers below each band represent fold change vs. NT shRNA group and normalized to actin as determined by scanning densitometry. Wild-type HMC-1 5C6 cells were left untreated (NT) or pretreated for one hour with 20 µM chloroquine (CQ) or 2 µM rapamycin (Rap). These cells, along with HMC-1 5C6 cells stably expressing NT, Atg5 or Atg7 specific shRNA were left uninfected, or infected at a 1∶20 MOI with <i>P. aeruginosa</i> strain 8821 for 3 hours. Cell impermeable antibiotics were added to kill extracellular bacteria for 3 hours. Cells were then lysed and subjected to Western blot analysis for the autophagy marker LC3 and acting loading control (B). Alternatively HMC1-5C6 cells expressing LC3-GFP and treated as above were monitored by laser scanning confocal microscopy (C and D) (n = 3 ± SEM, **p<0.01, ***p<0.005). Lysates were prepared from HMC-1 5C6 cells treated as described above, and incubated in the presence of cell impermeable antibiotic for 10 minutes to determine bacterial internalization (E) or 3 hours to examine bacterial killing (F). Serial dilutions of these lysates were streaked on LB agar plates and CFU/mL lysate was determined (n = 6± SEM, *p<0.05, **p<0.01).