FIG 8.

shRNA knockdown of macrophage PAP2a or lipin-1 reduced PGE₂ biosynthesis and COX-2 by F. tularensis-infected macrophages. (a) Steady-state mRNA was collected from wild-type (WT) RAW 264.7, knockdown, or nontarget shRNA cell lines, and knockdown was confirmed by RT-PCR. (b) RAW 264.7 knockdown or nontarget cell lines were inoculated with F. tularensis LVS at an MOI of 200:1. PGE₂ in the supernatant was quantified by enzyme-linked immunosorbent assay 24 hours after inoculation (n = 3). (c) Knockdown or nontarget cell lines were inoculated with F. tularensis LVS at an MOI of 200:1. Whole-cell lysates were collected 20 hours after inoculation, and COX-2 protein was quantified by Western blotting. Data are expressed as a fold change in COX-2 band intensity compared to its respective uninfected control (n = 4). (d) RAW 264.7 knockdown or nontarget cell lines were inoculated with F. tularensis LVS at an MOI of 200:1. TNF-α in the supernatant was quantified by enzyme-linked immunosorbent assay at 24 hours postinoculation (n = 3). (e) Knockdown or nontarget cell lines were inoculated with F. tularensis LVS at an MOI of 200:1. At 6 and 24 hours postinoculation, macrophages were lysed and intramacrophage bacteria were quantified (n = 3). An asterisk denotes a statistical difference compared to the nontarget (NT) shRNA-infected group (P ≤ 0.05). Graphs are means of pooled experiments ± SEMs.