Figure 2: Targeting negative regulatory nodes controlling oxidative burst enhances bactericidal activity in macrophages.

(a) Hits from the primary screen were validated and functionally characterized using the top two hairpins per gene, as defined by target gene knockdown efficiency in the primary screen (see Supplementary Data 1). Accordingly, RAW264.7 cells were subjected to shRNA knockdown followed by IFN-γ priming and zymosan or Staphylococcus aureus stimulation to induce oxidative burst. Data represent ROS production as relative luminescence units (RLU) measured with luminol substrate. (b) Target gene knockdown was measured by qPCR and normalized to Actb housekeeping gene or by western blot. Data represent mean±s.d. of biological triplicates. (c) The indicated genes were knocked down by shRNA in RAW264.7 cells. After IFN-γ priming, macrophages were infected with Staphylococcus aureus for 30 min, washed and cultured in gentamicin media to kill any bacteria that had not been phagocytosed. After the indicated time points, cells were lysed, and remaining live bacteria were enumerated as colony-forming units (c.f.u.). Data represent the mean of triplicates±s.d. *P≤0.05 as determined by Student’s t-test.